Cell-permeable peptide inhibitors of the JNK signal transduction pathway

ABSTRACT

The invention provides cell-permeable peptides that selectively block the branch of the JNK signaling pathway controlled by the islet-brain (IB) proteins. The provided cell-permeable peptides block the binding of intermediate kinases in the c-Jun amino terminal kinase (JNK) signaling pathway, thereby decreasing the downstream effects of c-Jun amino terminal kinase (JNK).

RELATED U.S. APPLICATION

[0001] This patent application claims priority to U.S. Ser. No.60/347,062, filed Jan. 9, 2002, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to protein kinase inhibitors andmore specifically to inhibitors of the protein kinase c-Jun aminoterminal kinase signal transduction pathway.

BACKGROUND OF THE INVENTION

[0003] The c-Jun amino terminal kinase (JNK) is a member of thestress-activated group of mitogen-activated protein (MAP) kinases. TheJNK signal transduction pathway is activated in response toenvironmental stress and by several classes of cell surface receptors,such as for example, cytokine receptors, serpentine receptors, andreceptor tyrosine kinases. JNK is activated by dual phosporylation thatis mediated by a protein kinase cascade that consists of a MAP kinase(MAPK), a MAP kinase kinase (MAPKK), and a MAP kinase kinase kinase(MAPKKK). Targets of the JNK signaling pathway include transcriptionsfactors, such as for example, the transcription factors ATF2 and c-Jun.

[0004] These kinases have been implicated in the control of cell growthand differentiation, and, more generally, in the response of cells toenvironmental stimuli. In mammalian cells, JNK has been implicated insuch biological processes as oncogenic transformation and in mediatingadaptive responses to environmental stress. JNK has also been associatedwith modulating immune responses, including maturation anddifferentiation of immune cells, as well effecting programmed cell deathin cells identified for destruction by the immune system.

[0005] Studies have implicated the JNK signaling pathway in apoptosisand survival signaling, and in particular, JNK has been recognized as acomponent of the stress-induced apoptotic signaling mechanism. Studieshave shown that JNK is required for the stress-induced release ofmitochondrial cytochrome c, and therefore, JNK is required forstress-induced apoptosis that is mediated by the mitochondrial/caspase-9pathway.

SUMMARY OF THE INVENTION

[0006] The present invention is based in part on the discovery ofcell-permeable peptides that selectively block the branch of the JNKsignaling pathway controlled by the islet-brain (IB) proteins (alsoreferred to as insulin binding (IB) proteins). The peptides, referred toherein as SH3 binding peptides (SH3-BP), block the binding ofintermediate kinases in the c-Jun amino terminal kinase (JNK) signalingpathway, thereby decreasing the downstream effects of c-Jun aminoterminal kinase (JNK).

[0007] Accordingly, the invention includes novel SH3 binding peptides,as well as chimeric peptides which include an SH3 binding peptide linkedto a trafficking peptide that can be used to direct a peptide on whichit is present to a desired cellular location. The trafficking sequencecan be used to direct transport of the peptide across the plasmamembrane. Alternatively, or in addition, the trafficking peptide can beused to direct the peptide to a desired intracellular location, such asthe nucleus.

[0008] In its various aspects, the invention includes an SH3 bindingpeptide having the amino acid sequence of SEQ ID NO: 1-35. The SH3binding peptide binds an islet-brain (IB) polypeptide, such as IB1 orIB2. Alternatively, the SH3 binding peptide inhibits the binding of anMKK7 kinase to an SH3 domain polypeptide. The SH3 binding peptide isless than 500 amino acids in length, e.g., less than or equal to 400,300, 200, 100, 50 or 25 amino acids in length.

[0009] In another aspect, the invention includes a chimeric peptidehaving a first domain and a second domain that are linked by a covalentbond, such that the first domain includes an amino acid sequence derivedfrom the human immunodeficiency virus (HIV) 1 TAT polypeptide and thesecond domain includes an SH3 binding peptide, e.g., SEQ ID NO: 1-35. Achimeric peptide includes for example SEQ ID NO: 3-4 and 20-21. In someaspects, the SH3 binding peptide binds an islet-brain (IB) polypeptide.

[0010] In another aspect, the invention includes a peptide having anSXSVGX (SEQ ID NO: 5) motif and a PPSPRP (SEQ ID NO: 6) motif, and bindsan SH3 domain polypeptide, such as an islet-brain (IB) polypeptide.Preferably, the peptide is less than 50 amino acids in length. In someaspects, the peptide includes the trafficking sequence of SEQ ID NO: 36.

[0011] The SH3 binding peptides can be present as polymers of L-aminoacids. Alternatively, the peptides can be present as polymers of D-aminoacids. In another embodiment, the peptides can be present asretro-inverso isomers of a peptide.

[0012] Also included in the invention are pharmaceutical compositionsthat include the SH3 binding peptides, as well as antibodies thatspecifically recognize the SH3 binding peptides.

[0013] In another aspect, the invention includes an insolated nucleicacid that encodes an SH3 binding peptide containing the amino acidsequence of SEQ ID NO: 1-35. The invention also includes a vectorcontaining the isolated nucleic acid that encodes an SH3 binding peptidecontaining the amino acid sequence of SEQ ID NO: 1-35, as well as a cellthat contains such a vector.

[0014] In another aspect, the invention includes a method of inhibitingapoptosis in a cell, e.g., a pancreatic cell or a neuronal cell, bycontacting the cell with an SH3 binding peptide of the invention, e.g.,SEQ ID NO: 2. The cell is a neuronal cell or a pancreatic cell. Inanother embodiment, the cell is contacted either in vitro, in vivo, orex vivo.

[0015] Also included in the invention is a method of alleviating asymptom of an apoptosis-associated disorder, e.g., a neurologicaldisorder, a neurodegenerative disorder, or a pancreatic disorder, in asubject by administering a SH3 binding peptide of the invention. Forexample, the subject is administered a polypeptide containing the aminoacid sequence of SEQ ID NO: 2.

[0016] In another aspect, the invention includes a method of promotingneuronal cell growth or regeneration by contacting a neuronal cell witha SH3 binding protein, e.g., SEQ ID NO: 2.

[0017] Among the advantages provided by the invention is that the SH3binding peptides are small, and can be produced readily in bulkquantities and in high purity. The binding peptides are also resistantto intracellular degradation, and are weakly immunogenic. Accordingly,the peptides are well suited for in vitro and in vivo applications inwhich inhibition of JNK-signaling is desired.

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0019] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a diagram showing alignments of amino acid sequencesthat bind the SH3 domain of IB1/2.

[0021]FIG. 2 is an illustration demonstrating the effects of IB1, IB2,IB1-TAT_((1 μM)), IB1-TAT-αSH3_((0.1 μM)), IB1-TAT-αSH3_((1 μM)),IB2-TAT-αSH3_((1 μM)) on MKK7 binding to IB1/2.

[0022]FIG. 3 is a histogram depicting inhibition of IL-1β-induced deathin insulin-secreting pancreatic β-cells by the TAT-αSH3 peptide.

[0023] FIGS. 4A-4F are illustrations demonstrating the effects ofJNK1_((1 μM)) and TAT-αSH3_((1 μM)) peptide on developing rat corticalneurons, as evidenced by the appearance of residual necrotic bodies(indicated as arrows). Panels B and C show the toxicity of JNK1_((1 μM))on developing rat cortical neurons, as compared to control developingrat cortical neuron shown in Panel A. Panel D shows control developingrat cortical neurons. Panels E and F show that the TAT-αSH3_((1 μM))peptide is not toxic to developing rat cortical neurons, as compared tocontrol developing rat cortical neurons shown in Panel D.

[0024]FIG. 5A is an illustration demonstrating the inhibition ofNMDA-induced death by the TAT-αSH3_((1 μM)) peptide in neurons stainedwith Hoechst/PI.

[0025]FIG. 5B is a histogram demonstrating the inhibition ofNMDA-induced death by the TAT-αSH3_((1 μM)) peptide in neurons stainedwith lactate dehydrogenase (LDH).

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention is based in part on the discovery of cellpermeable peptides that inhibit the branch of the activated c-Jun aminoterminal kinase (JNK) signaling pathway that is controlled by theislet-brain (IB) proteins (also referred to as insulin binding (IB)proteins). These cell-permeable peptides are referred to herein as SH3binding peptides (SH3-BP). Additionally, the discovery provides methodsof inhibiting apoptosis and methods and pharmaceutical compositions fortreating or alleviating a symptom of an apoptosis-associated disorders.The discovery further provides methods of promoting (i.e., increasing)neuronal cell growth and regeneration. By cell permeable it is meantthat the peptides are capable of crossing a biological membrane, such asa cellular or nuclear membrane.

[0027] Mitogen-activated protein kinase (MAPK) pathways, such as theextracellular-regulated kinases (ERKs)-1/2, p38 kinases, and the c-JunNH₂-terminal kinases (JNKs) signaling pathway, have a core unit formedby a three-member protein kinase cascade. Within the three-kinasemodule, the MAPK is phosphorylated and activated by MAPK kinases, knownas MKKs. Typically, the MKKs are dual specificity kinases that catalyzethe phosphorylation of MAPKs on both tyrosine and threonine residues. Inturn, the MKKs are phosphorylated and activated by serine/threoninekinases that function as MKK kinases, known as MKKKs. See Garrington, etal., Curr. Op. in Cell Biol. 11:211-218 (1991).

[0028] In the JNK signaling pathway, this protein kinase cascade isformed by three sequential kinases, known as MLK, MKK7 and JNK. Thesethree sequential kinases interact with, and are organized by, scaffoldproteins known as the insulin-binding, or islet-brain, (IB) proteins.The IB proteins are transcription factors that exhibit sequence-specificDNA binding activity. IB1 is a transcriptional activator that isinvolved in the control of the glucose transporter gene GLUT2 andinsulin genes, through interaction with homologous cis-regulatoryelements of the GLUT2 and insulin promoters. In particular, IB1 binds tothe GTII cis-element of the GLUT2 and insulin genes (see U.S. Pat. No.5,880,261). The IB proteins, and in particular, IB1 and IB2, areexpressed predominantly in the brain and pancreas. Accordingly, theSH3-BPs of the invention are useful in specifically targeting the JNKsignaling pathway in the brain and pancreas.

[0029] The intermediate kinase MKK7 of the three-kinase module binds tothe highly conserved Src-homology 3 (SH3) domain regions contained inthe IB1 and IB2 proteins. SH3 domains are small protein modulescontaining approximately 50 to 60 amino acid residues. These domainshave been identified in a variety of intracellular signaling andmembrane-associated polypeptides. The SH3 domain has been found tomediate protein-protein interactions that are involved in the couplingof intracellular signaling pathways, regulation of catalytic activity ofproteins, recruitment of substrates to enzymes, and localization ofproteins to a specific subcellular compartment. See Weng et al., Mol.and Cell. Biol. 15(10):5627-34 (1995).

[0030] The core, conserved binding motif of the SH3 domain isPro-x-x-Pro (SEQ ID NO: 35). SH3 domains generally bind to proline-richpeptides, thereby forming an extended, left-hand helical conformation,known as the polyproline-2 (PPII) helix. See Mayer, J. Cell Sci.114(7):1253-63 (2001).

[0031] The SH3 binding peptides of the invention were identified bypanning a phage display library against GST-SH3_(IB1/2) fusion proteinsto characterize peptides that bind to the highly conserved SH3 domainsof IB1 and IB2 (See, Example 1). SH3 binding peptides obtained from thebiopanning experiment are shown in FIG. 1.

[0032] Sequence comparison between the sequences obtained from the phagedisplay biopanning experiment, as shown in FIG. 1, revealed twoconserved 6 amino acid sequence motifs SXSVGX (SEQ ID NO: 5) and PPSPRP(SEQ ID NO: 6). The latter sequence corresponds to the SH3 bindingconsensus sequence, PXXP (SEQ ID NO: 35), shown in FIG. 1.

[0033] The SH3 binding peptides of the invention can be used in anysituation in which inhibition of JNK signaling is desired. This includesin vitro applications, ex vivo, and in vivo applications. As JNKs andall its isoforms participate in the development and establishment ofpathological states or in pathways, the SH3 binding peptides can be usedto prevent or inhibit the occurrence of such pathological states. Thisincludes prevention, treatment and alleviation of symptoms of diseasesand prevention, treatment and alleviation of symptoms of conditionssecondary to therapeutic actions. The SH3-BPs of the invention areuseful in treatment, prevention or alleviation of symptoms of pancreaticdisorders, neurodegenerative diseases and apoptotic associated disordersof for example the pancreas and the brain. For example, the peptides ofthe present invention can be used to treat or prevent or alleviate asymptom of, e.g., pancreatic disorders such as diabetes, pancreatitis,or pancreatic cancer; neurodegenerative and neurological disorders suchas Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease,Huntington's disease, Alzheimer's disease, schizophrenia and stroke;ionizing radiation; immune responses (including autoimmune diseases);ischemia/reperfusion injuries; heart and cardiovascular hypertrophies;and some cancers (e.g., Bcr-Abl transformation).

[0034] The SH3-BPs are also used to inhibit expression of genes and geneproducts whose expression increases in the presence of an active JNKpolypeptide, such as for example, proinflammatory cytokines.Proinflammatory cytokines are found in all forms of inflammatory,auto-inflammatory, immune and autoimmune diseases, degenerativediseases, myopathies, cardiomyopathies, and graft rejection.

[0035] The polynucleotides provided by the present invention are used toexpress recombinant peptides for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingpeptides is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates). Other uses for the nucleic acids include, e.g., molecularweight markers in gel electrophoresis-based analysis of nucleic acids.

[0036] The SH3 binding peptides disclosed herein are presented inTable 1. The table presents the name of the SH3 binding peptide, as wellas its sequence identifier number, length, and amino acid sequence. Theabbreviation “RV”, as used herein, refers to a “retro-inverso isomer” ofa peptide. TABLE 1 SEQ PEPTIDE NAME ID NO AA Sequence αSH3 1 12SVSVGMPPSP RP αSH3 (generic) 2 12 SX (S/P) V (G/L) XPPSP RP TAT-αSH3 321 RKKRRQRRRS VSVGMPPSPR P TAT-αSH3 4 29 XXXXRKKRRQ RRRXXXXSX (generic)(S/P) V (G/L) XPPSPRP αSH3 binding 5 6 SXSVGX motif 1 (generic) αSH3binding 6 6 PPSPRP motif 2 (generic) αSH3₂ 7 12 SVSVGMKPSP RP αSH3₃ 8 12SVSVGKNPSP RH αSH3₄ 9 12 TQPMMAPPSP RQ αSH3₅ 10 12 LDSLCHPQSP RP αSH3₆11 11 HPFLVSSSPR P αSH3₇ 12 9 GQPFFSPFS αSH3₈ 13 11 PPSNLIPPTL R αSH3₉14 6 SPPSNL αSH3₁₀ 15 11 FNPWSSKPSL L αSH3₁₁ 16 12 NASVGNDHSH SH αSH3₁₂17 11 EHMALTYPFR P RV-αSH3 18 12 PRPSPPMGVS VS RV-αSH3 (generic) 19 12PRPSPPX (G/L) V (S/P) XS RV-TAT-αSH3 20 21 PRPSPPMGVS VSRRRQRRKK RRV-TAT-αSH3 21 29 PRPSPPX (G/L) V (S/P) (generic) XSXXXXRRRQ RRKKRXXXXRV-αSH3 binding 22 6 XGVSXS motif 1 (generic) RV-αSH3 binding 23 6PRPSPP motif 2 (generic) RV-αSH3₂ 24 12 PRPSPKMGVS VS RV-αSH3₃ 25 12HRPSPNKGVS VS RV-αSH3₄ 26 12 QRPSPPAMMP QT RV-αSH3₅ 27 12 PRPSQPHCLS DLRV-αSH3₆ 28 11 PRPSSSVLFP H RV-αSH3₇ 29 9 SFPSFFPQG RV-αSH3₈ 30 11RLTPPILNSP P RV-αSH3₉ 31 6 LNSPPS RV-αSH3₁₀ 32 11 LLSPKSSWPN F RV-αSH3₁₁33 12 HSHSHDNGVS AN RV-αSH3₁₂ 34 11 PRFPYTLAMH E

[0037] SH3 Binding Peptides

[0038] In one aspect, the invention provides an SH3 binding peptide.Exemplary SH3 binding peptides include the amino acid sequences of SEQID NO: 1-35. No particular length is implied by the term “peptide.” Insome embodiments, the SH3 binding peptide is less than 500 amino acidsin length, e.g., less than or equal to 450, 400, 350, 300, 250, 200,150, 100, 75, 50, 35, or 25 amino acids in length. Preferably, thepeptide is capable of transport across a biological membrane, e.g., anuclear or cellular membrane. In various embodiment, the SH3 bindingpeptide includes the amino acid sequence of one or more of SEQ ID NO:1-35. The SH3 binding peptides bind at least one IB protein, e.g., IB1or IB2. Binding of at least one IB protein, e.g., IB1 or IB2, can bemeasured by methods known in the art, such as for example, by using anaffinity binding assay having a GST-IB1 or GST-IB2 fusion protein as anaffinity matrix. Alternatively, the peptide inhibits MKK7 binding to anSH3 domain polypeptide. Inhibition of MKK7 binding is measured bymethods known in the art, for example using pull down experiments asdescribed in Example 3. Inhibition of MKK7 binding is also measured byobserving inhibition of JNK signaling, i.e., JNK activation. JNKsignaling is determined for example using a solid phase JNK assay asdescribed herein (see e.g., Example 7) and in Bonny et al., Diabetes50:77-82 (2001). The term “an SH3 domain polypeptide”, as used herein,is meant to refer to a polypeptide that contains one or more SH3 domainconsensus sequences. A SH3 consensus sequence as described by Pfamdatabase entry pfam00018.6 includes the amino acid sequencePKVVALYDYQARE-SDELSFK-KGDIIIVLEKSDD—GWWKGRLKGT—KEGLIPSNYVEPV (SEQ ID NO:40). Exemplary, SH3 polypeptides include intracellular signalingproteins such as the Src, Abl, Csk and ZAP70 families of proteintyrosine kinases (e.g., GenBank Accession No. P12931, P00519, P41240 andP43403, respectively, incorporated herein by reference in theirentirety); mammalian phosphatidylinositol-specific phospholipase C-γ-1and C-γ-2 (e.g., GenBank Accession No. NP_(—)002652 and NP_(—)002651,respectively, incorporated herein by reference in their entirety);mammalian phosphatidylinositol 3-kinase regulatory p85 subunit (e.g.,GenBank Accession No: A38748, incorporated herein by reference in itsentirety); mammalian Ras GTPase-activating protein (GAP) (e.g., GenBankAccession No: BAA11230, incorporated herein by reference in itsentirety); adaptor proteins that mediate binding of guanine nucleotideexchange factors to growth factor receptors such as vertebrate GRB2(e.g., GenBank Accession No: P29354 and AAC72075, incorporated herein byreference in their entirety); and cytoskeletal proteins such as fodrin(e.g., GenBank Accession No. AAA51702, AAA52468 and AAB28324,incorporated herein by reference in their entirety) and yeast actinbinding protein ABP-1 (e.g., GenBank Accession No. LLBY, incorporatedherein by reference in its entirety).

[0039] Examples of SH3 binding peptides include a peptide which includes(in whole or in part) the sequences of SEQ ID NO: 1-35 as shown inTable 1. As used herein, X may be any amino acid. The single residuerepresented by (S/P) may be either Ser or Pro in the generic sequence.The single residue represented by (G/L) may be either Gly or Leu in thegeneric sequence.

[0040] The SH3 binding peptides can be polymers of L-amino acids,D-amino acids, or a combination of both. For example, in variousembodiments, the peptides are D retro-inverso peptides. The term“retro-inverso isomer” refers to an isomer of a linear peptide in whichthe direction of the sequence is reversed, and the term “D-retro-inversoisomer” refers to an isomer of a linear peptide in which the directionof the sequence is reversed and the chirality of each amino acid residueis inverted. See, e.g., Jameson et al., Nature, 368, 744-746 (1994);Brady et al., Nature, 368, 692-693 (1994). The net result of combiningD-enantiomers and reverse synthesis is that the positions of carbonyland amino groups in each amide bond are exchanged, while the position ofthe side-chain groups at each alpha carbon is preserved. Unlessspecifically stated otherwise, it is presumed that any given L-aminoacid sequence of the invention may be made into an D retro-inversopeptide by synthesizing a reverse of the sequence for the correspondingnative L-amino acid sequence.

[0041] SH3 binding peptides may be obtained or produced by methodswell-known in the art, e.g chemical synthesis, genetic engineeringmethods as discussed below. For example, a peptide corresponding to aportion of an SH3 binding peptide including a desired region or domain,or that mediates the desired activity in vitro, may be synthesized byuse of a peptide synthesizer.

[0042] A candidate SH3 binding peptide may also be analyzed byhydrophilicity analysis (see, e.g., Hopp and Woods, 1981. Proc Natl AcadSci USA 78: 3824-3828) that can be utilized to identify the hydrophobicand hydrophilic regions of the peptides, thus aiding in the design ofsubstrates for experimental manipulation, such as in bindingexperiments, antibody synthesis. Secondary structural analysis may alsobe performed to identify regions of an SH3 binding peptide that assumespecific structural motifs. See e.g., Chou and Fasman, 1974. Biochem 13:222-223. Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies can beaccomplished using computer software programs available in the art.Other methods of structural analysis including, e.g., X-raycrystallography (see, e.g., Engstrom, 1974. Biochem Exp Biol 11: 7-13);mass spectroscopy and gas chromatography (see, e.g., Methods in ProteinScience, 1997. J. Wiley and Sons, New York, N.Y.) and computer modeling(see, e.g., Fletterick and Zoller, eds., 1986. Computer Graphics andMolecular Modeling, In: Current Communications in Molecular Biology,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) may alsobe employed.

[0043] The present invention additionally relates to nucleic acids thatencode SH3 binding peptides having L-form amino acids, e.g., thoseL-peptides indicated in Table 1, as well as the complements of thesesequences. Nucleic acids encoding the SH3 binding peptides may beobtained by any method known in the art (e.g, by PCR amplification usingsynthetic primers hybridizable to the 3′- and 5′-termini of the sequenceand/or by cloning from a cDNA or genomic library using anoligonucleotide sequence specific for the given gene sequence).

[0044] For recombinant expression of one or more SH3 binding peptides,the nucleic acid containing all or a portion of the nucleotide sequenceencoding the peptide may be inserted into an appropriate expressionvector (i e., a vector that contains the necessary elements for thetranscription and translation of the inserted peptide coding sequence).In some embodiments, the regulatory elements are heterologous (i.e., notthe native gene promoter). Alternately, the necessary transcriptionaland translational signals may also be supplied by the native promoterfor the genes and/or their flanking regions.

[0045] A variety of host-vector systems may be utilized to express thepeptide coding sequence(s). These include, but are not limited to: (i)mammalian cell systems that are infected with vaccinia virus,adenovirus, and the like; (ii) insect cell systems infected withbaculovirus and the like; (iii) yeast containing yeast vectors or (iv)bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. Depending upon the host-vector system utilized, any one of a numberof suitable transcription and translation elements may be used.

[0046] Promoter/enhancer sequences within expression vectors may utilizeplant, animal, insect, or fungus regulatory sequences, as provided inthe invention. For example, promoter/enhancer elements can b used fromyeast and other fungi (e.g., the GAL4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter). Alternatively, or in addition, they mayinclude animal transcriptional control regions, e.g., (i) the insulingene control region active within pancreatic β-cells (see, e.g.,Hanahan, et al., 1985. Nature 315: 115-122); (ii) the immunoglobulingene control region active within lymphoid cells (see, e.g., Grosschedl,et al., 1984. Cell 38: 647-658); (iii) the albumin gene control regionactive within liver (see, e.g., Pinckert, et al., 1987. Genes and Dev 1:268-276; (iv) the myelin basic protein gene control region active withinbrain oligodendrocyte cells (see, e.g., Readhead, et al., 1987. Cell 48:703-712); and (v) the gonadotropin-releasing hormone gene control regionactive within the hypothalamus (see, e.g., Mason, et al., 1986. Science234: 1372-1378), and the like.

[0047] Expression vectors or their derivatives include, e.g. human oranimal viruses (e.g., vaccinia virus or adenovirus); insect viruses(e.g., baculovirus); yeast vectors; bacteriophage vectors (e.g., lambdaphage); plasmid vectors and cosmid vectors.

[0048] A host cell strain may be selected that modulates the expressionof inserted sequences of interest, or modifies or processes expressedpeptides encoded by the sequences in the specific manner desired. Inaddition, expression from certain promoters may be enhanced in thepresence of certain inducers in a selected host strain; thusfacilitating control of the expression of a genetically-engineeredpeptides. Moreover, different host cells possess characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation, andthe like) of expressed peptides. Appropriate cell lines or host systemsmay thus be chosen to ensure the desired modification and processing ofthe foreign peptide is achieved. For example, peptide expression withina bacterial system can be used to produce an unglycosylated corepeptide; whereas expression within mammalian cells ensures “native”glycosylation of a heterologous peptide.

[0049] Also included in the invention are derivatives, fragments,homologs, analogs and variants of SH3 binding peptides and nucleic acidsencoding these peptides. For nucleic acids, derivatives, fragments, andanalogs provided herein are defined as sequences of at least 6(contiguous) nucleic acids, and which have a length sufficient to allowfor specific hybridization. For amino acids, derivatives, fragments, andanalogs provided herein are defined as sequences of at least 4(contiguous) amino acids, a length sufficient to allow for specificrecognition of an epitope.

[0050] The length of the fragments are less than the length of thecorresponding full-length nucleic acid or polypeptide from which the SH3binding peptide, or nucleic acid encoding same, is derived. Derivativesand analogs may be full length or other than full length, if thederivative or analog contains a modified nucleic acid or amino acid.Derivatives or analogs of the SH3 binding peptides include, e.g.,molecules including regions that are substantially homologous to thepeptides, in various embodiments, by at least about 30%, 50%, 70%, 80%,or 95%, 98%, or even 99%, identity over an amino acid sequence ofidentical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art. Forexample sequence identity can be measured using sequence analysissoftware (Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), with the default parameters therein.

[0051] In the case of polypeptide sequences, which are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine. Thus, included in the invention are peptides havingmutated sequences such that they remain homologous, e.g. in sequence, infunction, and in antigenic character or other function, with a proteinhaving the corresponding parent sequence. Such mutations can, forexample, be mutations involving conservative amino acid changes, e.g.,changes between amino acids of broadly similar molecular properties. Forexample, interchanges within the aliphatic group alanine, valine,leucine and isoleucine can be considered as conservative. Sometimessubstitution of glycine for one of these can also be consideredconservative. Other conservative interchanges include those within thealiphatic group aspartate and glutamate; within the amide groupasparagine and glutamine; within the hydroxyl group serine andthreonine; within the aromatic group phenylalanine, tyrosine andtryptophan; within the basic group lysine, arginine and histidine; andwithin the sulfur-containing group methionine and cysteine. Sometimessubstitution within the group methionine and leucine can also beconsidered conservative. Preferred conservative substitution groups areaspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine;alanine-valine; phenylalanine-tyrosine; and lysine-arginine.

[0052] Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Of course, other polypeptides willmeet the same criteria.

[0053] The invention also encompasses allelic variants of the disclosedpolynucleotides or peptides; that is, naturally-occurring alternativeforms of the isolated polynucleotide that also encode peptides that areidentical, homologous or related to that encoded by the polynucleotides.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

[0054] Species homologs of the disclosed polynucleotides and peptidesare also provided by the present invention. “Variant” refers to apolynucleotide or polypeptide differing from the polynucleotide orpolypeptide of the present invention, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and in manyregions, identical to the polynucleotide or polypeptide of the presentinvention. The variants may contain alterations in the coding regions,non-coding regions, or both.

[0055] In some embodiments, altered sequences include insertions suchthat the overall amino acid sequence is lengthened while the proteinretains trafficking properties. Additionally, altered sequences mayinclude random or designed internal deletions that shorten the overallamino acid sequence while the protein retains transport properties.

[0056] The altered sequences can additionally or alternatively beencoded by polynucleotides that hybridize under stringent conditionswith the appropriate strand of the naturally-occurring polynucleotideencoding a polypeptide or peptide from which the SH3 binding peptide isderived. The variant peptide can be tested for IB-binding and modulationof JNK-mediated activity using the herein described assays. ‘Stringentconditions’ are sequence dependent and will be different in differentcircumstances. Generally, stringent conditions can be selected to beabout 5° C. lower than the thermal melting point (T_(M)) for thespecific sequence at a defined ionic strength and pH. The T_(M) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

[0057] High stringency can include, e.g., Step 1: Filters containing DNAare pretreated for 8 hours to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 48 hours at 65° C. in the above prehybridization mixtureto which is added 100 mg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Step 3: Filters are washed for 1 hour at 37° C. ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Step 4:Filters are autoradiographed. Other conditions of high stringency thatmay be used are well known in the art. See, e.g., Ausubel et al.,(eds.), 1993, Current Protocols in Molecular Biology, John Wiley andSons, NY; and Kriegler, 1990, Gene Transfer and Expression, a LaboratoryManual, Stockton Press, NY.

[0058] Moderate stringency conditions can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100mg/ml denatured salmon sperm DNA. Step 2: Filters are hybridized for18-20 hours at 55° C. in the same solution with 5-20×106 cpm ³²P-labeledprobe added. Step 3: Filters are washed at 37° C. for 1 hour in asolution containing 2×SSC, 0.1% SDS, then washed twice for 30 minutes at60° C. in a solution containing 1×SSC and 0.1% SDS. Step 4: Filters areblotted dry and exposed for autoradiography. Other conditions ofmoderate stringency that may be used are well-known in the art. See,e.g., Ausubel et al., (eds.), 1993, Current Protocols in MolecularBiology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY.

[0059] Low stringency can include: Step 1: Filters containing DNA arepretreated for 6 hours at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 18-20 hours at 40° C. in the same solution with theaddition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm ³²P-labeled probe.Step 3: Filters are washed for 1.5 hours at 55° C. in a solutioncontaining 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Thewash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Step 4: Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and reexposed to film. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations). See, e.g., Ausubel et al., (eds.),1993, Current Protocols in Molecular Biology, John Wiley and Sons, NY;and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY.

[0060] Chimeric Peptides Including an SH3 Binding Domain and aTrafficking Domain

[0061] In another aspect the invention provides a chimeric peptide thatincludes a first and second domain. The first domain includes atrafficking sequence, while the second domain includes an SH3 bindingpeptide linked by a covalent bond, e.g. peptide bond, to the firstdomain. The first and second domains can occur in any order in thepeptide, and the peptide can include one or more of each domain.

[0062] A trafficking sequence is any sequence of amino acids thatdirects a peptide in which it is present to a desired cellulardestination. Thus, the trafficking sequence can direct the peptideacross the plasma membrane, e.g., from outside the cell, through theplasma membrane, and into the cytoplasm. Alternatively, or in addition,the trafficking sequence can direct the peptide to a desired locationwithin the cell, e.g., the nucleus, the ribosome, the ER, a lysosome, orperoxisome.

[0063] In some embodiments, the trafficking peptide is derived from aknown membrane-translocating sequence. For example, the traffickingpeptide may include sequences from the human immunodeficiency virus(HIV)1 TAT protein. This protein is described in, e.g., U.S. Pat. Nos.5,804,604 and 5,674,980, each incorporated herein by reference. The SH3binding peptide may be linked to some or all of the entire 86 aminoacids that make up the TAT protein. For example, a functionallyeffective fragment or portion of a TAT protein that has fewer than 86amino acids, which exhibits uptake into cells, and optionally uptakeinto the cell nucleus, can be used. In one embodiment, the fragmentincludes a peptide containing TAT residues 49-57, e.g.NH₂-RKKRRQRRR-COOH (SEQ ID NO: 36) or a generic TAT sequenceNH₂-X_(n)-RKKRRQRRR-X_(n)-COOH (SEQ ID NO: 37). A TAT peptide thatincludes the region that mediates entry and uptake into cells can befurther defined using known techniques. See, e.g., Franked et al., Proc.Natl. Acad. Sci, USA 86: 7397-7401 (1989).

[0064] The TAT sequence may be linked either to the N-terminal or theC-terminal end of the SH3 binding peptide. A hinge of two prolineresidues may be added between the TAT and SH3 binding peptide to createthe full fusion peptide. For example, amino acid fusion peptides may bethe TAT-αSH3 peptide (SEQ ID NO: 3) or the generic TAT-αSH3 peptide (SEQID NO: 4). Retro-inverso fusion peptides may be the RV-TAT-αSH3 peptide(SEQ ID NO: 20) or the generic RV-TAT-αSH3 peptide (SEQ ID NO: 21). TheTAT peptide may be a retro-inverso peptide having the sequenceNH₂-X_(n)-RRRQRRKKR-X_(n)-COOH (SEQ ID NO: 38) or the TAT-peptide can bea generic retro-inverso peptide having the sequenceNH₂-X_(n)-RRRQRRKKR-X_(n)-COOH (SEQ ID NO: 39). In SEQ ID NO: 3-4 and38-39, the number of “X” residues is not limited to the one depicted noris the number of Xs in a given peptide limited to the one depicted, andaccordingly, the “X” residues may vary as described above. The fusionpeptide can include one or more of the SH3-BPs of SEQ ID NO: 1-35. Forexample, the fusion peptide can be a chimeric peptide comprising thesequence of SEQ ID NO: 36 covalently linked to the sequence of SEQ IDNO: 7, or alternatively the chimeric peptide can comprise the sequenceof SEQ ID NO: 38 covalently linked to the sequence of SEQ ID NO: 24. Forexample, the fusion peptide can include a chimeric peptide comprisingthe sequence of SEQ ID NO: 36 covalently linked to a sequence selectedfrom SEQ ID NO: 7-17, or alternatively, the chimeric peptide cancomprise the sequence of SEQ ID NO: 38 covalently linked to an aminoacid sequence selected from the group consisting of SEQ ID NO: 24-34.Any combination of SH3 binding peptides and trafficking sequences arewithin the scope of the present invention.

[0065] The fusion peptide can also include a peptide comprising theamino acid sequence of SEQ ID NO: 36 covalently linked to a peptide thatincludes an SXSVGX (SEQ ID NO: 5) motif and a PPSPRP (SEQ ID NO: 6)motif. Alternatively, the fusion peptide may be a chimeric peptide thatincludes the amino acid sequence of SEQ ID NO: 38 covalently linked to apeptide containing an XGVSXS (SEQ ID NO: 22) and a PRPSPP (SEQ ID NO:23) motif. In one embodiment, the fusion peptide has a length that isless than 50 amino acids.

[0066] The trafficking sequence can be a single (i.e., continuous) aminoacid sequence present in the TAT sequence. Alternatively it can be twoor more amino acid sequences, which are present in TAT protein, but inthe naturally-occurring protein are separated by other amino acidsequences. As used herein, TAT protein includes a naturally-occurringamino acid sequence that is the same as that of naturally-occurring TATprotein, or its functional equivalent protein or functionally equivalentfragments thereof (peptides). Such functional equivalent proteins orfunctionally equivalent fragments possess uptake activity into the celland into the cell nucleus that is substantially similar to that ofnaturally-occurring TAT protein. TAT protein can be obtained fromnaturally-occurring sources or can be produced using genetic engineeringtechniques or chemical synthesis.

[0067] The amino acid sequence of naturally-occurring HIV TAT proteincan be modified, for example, by addition, deletion and/or substitutionof at least one amino acid present in the naturally-occurring TATprotein, to produce modified TAT protein (also referred to herein as TATprotein). Modified TAT protein or TAT peptide analogs with increased ordecreased stability can be produced using known techniques. In someembodiments TAT proteins or peptides include amino acid sequences thatare substantially similar, although not identical, to that ofnaturally-occurring TAT protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to TAT protein toproduce a modified TAT having increased membrane solubility.

[0068] Variants of the TAT protein can be designed to modulateintracellular localization of TAT-SH3 binding peptide. When addedexogenously, such variants are designed such that the ability of TAT toenter cells is retained (i.e., the uptake of the variant TAT protein orpeptide into the cell is substantially similar to that ofnaturally-occurring HIV TAT). For example, alteration of the basicregion thought to be important for nuclear localization (see, e.g., Dangand Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauber et al., J.Virol. 63: 1181-1187 (1989); Ruben et al., J. Virol. 63: 1-8 (1989)) canresult in a cytoplasmic location or partially cytoplasmic location ofTAT, and therefore, of the SH3 binding peptide. Alternatively, asequence for binding a cytoplasmic or any other component or compartment(e.g., endoplasmic reticule, mitochondria, gloom apparatus, lysosomalvesicles,) can be introduced into TAT in order to retain TAT and the SH3binding peptide in the cytoplasm or any other compartment to conferregulation upon uptake of TAT and the SH3 binding peptide.

[0069] Other sources for the trafficking peptide are well known in theart. For example, VP22 (described in, e.g., WO 97/05265; Elliott andO'Hare, Cell 88: 223-233 (1997)), or non-viral proteins (Jackson et al,Proc. Natl. Acad. Sci. USA 89: 10691-10695 (1992)). Other suitabletrafficking peptides include peptides derived from the Drosophilamelanogaster antennapedia (Antp) homeotic transcription factor, the hregion of the signal sequence of Kaposi fibroblast growth factor (MTS),and the protein PreS2 of hepatitis B virus (HBV) (Kelemen, et al., J.Biol. Chem. 277(10):8741-8748 (2002)). Alternatively, the traffickingpeptide is a polymer of cationic macromolecules or an arginine-richpeptide including a poly-arginine repeat.

[0070] The SH3 binding peptide and the trafficking sequence can belinked by chemical coupling in any suitable manner known in the art.Many known chemical cross-linking methods are non-specific, i.e.; theydo not direct the point of coupling to any particular site on thetransport polypeptide or cargo macromolecule. As a result, use ofnon-specific cross-linking agents may attack functional sites orsterically block active sites, rendering the conjugated proteinsbiologically inactive.

[0071] One way to increasing coupling specificity is to directlychemical coupling to a functional group found only once or a few timesin one or both of the polypeptides to be cross-linked. For example, inmany proteins, cysteine, which is the only protein amino acid containinga thiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

[0072] Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

[0073] Coupling of the two constituents can be accomplished via acoupling or conjugating agent. There are several intermolecularcross-linking reagents which can be utilized, See for example, Means andFeeney, Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43.Among these reagents are, for example, J-succinimidyl3-(2-pyridyldithio) propionate (SPDP) or N, N′-(1,3-phenylene)bismaleimide (both of which are highly specific for sulfhydryl groupsand form irreversible linkages); N, N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges (whichrelatively specific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages with amino andtyrosine groups). Other cross-linking reagents useful for this purposeinclude: p,p′-difluoro-m,m′-dinitrodiphenylsulfone (which formsirreversible cross-linkages with amino and phenolic groups); dimethyladipimidate (which is specific for amino groups);phenol-1,4-disulfonylchloride (which reacts principally with aminogroups); hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate (which reacts principally with amino groups);glutaraldehyde (which reacts with several different side chains) anddisdiazobenzidine (which reacts primarily with tyrosine and histidine).

[0074] Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

[0075] Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog ofMBS. The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

[0076] Cross-linking reagents often have low solubility in water. Ahydrophilic moiety, such as a sulfonate group, may be added to thecross-linking reagent to improve its water solubility. Sulfo-MBS andsulfo-SMCC are examples of cross-linking reagents modified for watersolubility.

[0077] Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

[0078] Numerous cross-linking reagents, including the ones discussedabove, are commercially available. Detailed instructions for their useare readily available from the commercial suppliers. A general referenceon protein cross-linking and conjugate preparation is: Wong, Chemistryof Protein Conjugation and Cross-Linking, CRC Press (1991).

[0079] Chemical cross-linking may include the use of spacer arms. Spacerarms provide intramolecular flexibility or adjust intramoleculardistances between conjugated moieties and thereby may help preservebiological activity. A spacer arm may be in the form of a polypeptidemoiety that includes spacer amino acids, e.g. proline. Alternatively, aspacer arm may be part of the cross-linking reagent, such as in“long-chain SPDP” (Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

[0080] Alternatively, the chimeric peptide can be produced as a fusionpeptide that includes the trafficking sequence and the SH3 bindingpeptide which can conveniently be expressed in known suitable hostcells. Fusion peptides, as described herein, can be formed and used inways analogous to or readily adaptable from standard recombinant DNAtechniques, as describe above.

[0081] Production of Antibodies Specific for SH3 Binding Peptides

[0082] SH3 binding peptides, including chimeric peptides including theSH3 binding peptides (e.g., peptides including the amino acid sequencesshown in Table 1), as well peptides, or derivatives, fragments, analogsor homologs thereof, may be utilized as immunogens to generateantibodies that immunospecifically-bind these peptide components. Suchantibodies include, e.g., polyclonal, monoclonal, chimeric, singlechain, Fab fragments and a Fab expression library. In a specificembodiment, antibodies to human peptides are disclosed. In anotherspecific embodiment, fragments of the SH3 binding peptides are used asimmunogens for antibody production. Various procedures known within theart may be used for the production of polyclonal or monoclonalantibodies to an SH3 binding peptide, or derivative, fragment, analog orhomolog thereof.

[0083] For the production of polyclonal antibodies, various host animalsmay be immunized by injection with the native peptide, or a syntheticvariant thereof, or a derivative of the foregoing.

[0084] Various adjuvants may be used to increase the immunologicalresponse and include, but are not limited to, Freund's (complete andincomplete), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, dinitrophenol, etc.) and human adjuvants such as BacilleCalmette-Guerin and Corynebacterium parvum.

[0085] For preparation of monoclonal antibodies directed towards an SH3binding peptide, or derivatives, fragments, analogs or homologs thereof,any technique that provides for the production of antibody molecules bycontinuous cell line culture may be utilized. Such techniques include,but are not limited to, the hybridoma technique (see, Kohler andMilstein, 1975. Nature 256: 495-497); the trioma technique; the humanB-cell hybridoma technique (see, Kozbor, et al., 1983. Immunol Today 4:72) and the EBV hybridoma technique to produce human monoclonalantibodies (see, Cole, et al., 1985. In: Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized in the practice of the present invention andmay be produced by the use of human hybridomas (see, Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cellswith Epstein Barr Virus in vitro (see, Cole, et al., 1985. In:Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., pp.77-96).

[0086] According to the invention, techniques can be adapted for theproduction of single-chain antibodies specific to an SH3 binding peptide(see, e.g., U.S. Pat. No. 4,946,778). In addition, methodologies can beadapted for the construction of Fab expression libraries (see, e.g.,Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor an SH3 binding peptide or derivatives, fragments, analogs orhomologs thereof. Non-human antibodies can be “humanized” by techniqueswell known in the art. See e.g., U.S. Pat. No. 5,225,539. Antibodyfragments that contain the idiotypes to an SH3 binding peptide may beproduced by techniques known in the art including, e.g., (i) an F(ab′)₂fragment produced by pepsin digestion of an antibody molecule; (ii) anFab fragment generated by reducing the disulfide bridges of an F(ab′)₂fragment; (iii) an Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) Fvfragments.

[0087] In one embodiment, methodologies for the screening of antibodiesthat possess the desired specificity include, but are not limited to,enzyme-linked immunosorbent assay (ELISA) and otherimmunologically-mediated techniques known within the art. In a specificembodiment, selection of antibodies that are specific to a particulardomain of an SH3 binding peptide is facilitated by generation ofhybridomas that bind to the fragment of an SH3 binding peptidepossessing such a domain. Antibodies that are specific for a domainwithin an SH3 binding peptide, or derivative, fragments, analogs orhomologs thereof, are also provided herein.

[0088] The anti-SH3 binding peptide antibodies may be used in methodsknown within the art relating to the localization and/or quantitation ofan SH3 binding peptide (e.g., for use in measuring levels of the peptidewithin appropriate physiological samples, for use in diagnostic methods,for use in imaging the peptide, and the like). In a given embodiment,antibodies for the SH3 binding peptides, or derivatives, fragments,analogs or homologs thereof that contain the antibody derived bindingdomain, are utilized as pharmacologically active compounds (hereinafter“Therapeutics”).

[0089] Methods of Inhibiting Apoptosis

[0090] Also included in the invention are methods for inhibitingapoptosis in a cell, treating an apoptosis-associated disorder oralleviating a symptom of an apoptosis-associated disorder in a subject.Apoptosis, also known as programmed cell death, plays a role indevelopment, aging and in various pathologic conditions.

[0091] An apoptosis associated disorder includes for example,immunodeficiency diseases, including AIDS/HIV, senescence, pancreaticdisorders such as diabetes (i.e., Type I or Type II), pancreatitis, andpancreatic cancer, neurological disorders (e.g., neurodegenerativediseases such as Amyotrophic Lateral Sclerosis, Parkinson's disease,Huntington's disease, Alzheimer's disease, and stroke, any degenerativedisorder, schizophrenia, ischemic and reperfusion cell death, acuteischemic injury, infertility, wound-healing, and the like.

[0092] Neurodegenerative diseases are characterized by gradualprogressive neuronal cell death. Other neurological disorders includeneuropathy, e.g., diabetic neuropathy, encephalitis and meningitis.Neurological disorders are diagnosed, typically by a physician usingstandard methodologies known be those skilled in the art. Such methodsinclude, neurologic history, neurological examination. Neurologicalexamination is accomplished by a systematic physical examination of allfunctions of the cerebrum, peripheral nerves and muscle. Diagnosis isalso made using techniques for imaging the nervous system with such ascomputed tomography, magnetic resonance imaging, myelography, andpositron emission tomography.

[0093] Some pancreatic disorders, e.g., diabetes or pancreatitis arecharacterized by gradual progressive pancreatic cell death. For example,in diabetes, insulin producing cells ( e.g., β-cells) are destroyedresulting in an insulin deficiency. In pancreatitis, local and systemicinflammation results in the release of cytokines which lead topancreatic cell death. Pancreatic disorders are diagnosed, typically bya physician using standard methodologies known be those skilled in theart. Such methods include elevated of serum amylase and lipase levels,hyperglycemia, hypocalcemia, or hyperbilirubinemia.

[0094] A symptom associated with an apoptosis-associated disorder ismeant to include any sensation or change in bodily function experiencedby a patient that is associated with a particular disease. For example,in diabetes, a symptom associated with the disorder includes low seruminsulin levels, high serum glucose, pancreatic β-cell death, neuropathy,and ketoacidosis. Alternatively, in a neurodegenerative disorder, asymptom includes, for example, neuronal cell death, neuron degeneration,neuron dysfunction, cerebral atrophy, accumulation of amyloid plaques inthe brain, and accumulation of filamentous structures (e.g., Lewybodies, taurich intraneuronal neurofibrillary tangles (NFTs)).

[0095] Many methods for measuring apoptosis, including those describedherein, are known to the skilled artisan including, but not limited to,the classic methods of DNA ladder formation by gel electrophoresis andof morphologic examination by electron microscopy. The more recent andreadily used method for measuring apoptosis is flow cytometry. Flowcytometry permits rapid and quantitative measurements on apoptoticcells. Many different flow cytometric methods for the assessment ofapoptosis in cells have been described (Darzynkiewicz et al. Cytometry13: 795-808, 1992). Most of these methods measure apoptotic changes incells by staining with various DNA dyes (i.e. propidium iodide (PI),DAPI, Hoechst 33342), however, techniques using the terminaldeoxynucleotidyl transferase (TUNNEL) or nick translation assays havealso been developed (Gorczyca et al. Cancer Res 53: 1945-1951, 1993).Recently, rapid flow cytometric staining methods that use Annexin V fordetection of phosphatidylserine exposure on the cell surface as a markerof apoptosis have become commercially available.

[0096] Apoptosis is inhibited in a cell by contacting a cell with an SH3binding peptide, an SH3 chimeric peptide, or nucleic acid encoding anSH3 binding peptide in an amount sufficient to inhibit apoptosis. Forexample the cell is contacted with any one of SEQ ID NO: 1-35. The cellis a pancreatic cell, e.g., a pancreatic β-cell or a neuronal cell. Thecell population that is exposed to, i.e., contacted with, the SH3binding peptide can be any number of cells, i.e., one or more cells, andcan be provided in vitro, in vivo, or ex vivo.

[0097] For example, to determine whether a compound inhibits cell death,a compound is tested by incubating the compound with a primary orimmortalized cell, inducing a state of oxidative stress of the cells(e.g., by incubating them with H₂O₂) and measuring cell viability bystandard methods. As a control the cells are incubated in the absence ifthe compound and then the treated cells are incubated in the absence ofthe compound and then treated to induce a state of oxidative stress. Adecrease in cell death (or an increase in the number of viable cells) inthe compound treated sample indicates that the compound inhibitsoxidative-stress induced cell death, i.e., apoptosis. The test isrepeated using different does of the compound to determine the doserange in which the compound functions to inhibit apoptosis.

[0098] An apoptosis-associated disorder is treated or a symptom ofapoptosis is alleviated in a subject by administering to a subject inneed thereof a biologically-active therapeutic compound (hereinafter“Therapeutic”).

[0099] The Therapeutics include, e.g.: (i) any one or more of the SH3binding peptides or SH3 chimeric peptides, and derivative, fragments,analogs and homologs thereof; (ii) antibodies directed against the SH3binding peptides; (iii) nucleic acids encoding an SH3 binding peptide orSH3 chimeric peptide, and derivatives, fragments, analogs and homologsthereof; (iv) antisense nucleic acids to sequences encoding an SH3binding peptide, and (v) modulators (i e., inhibitors, agonists andantagonists). For example, the therapeutic includes SEQ ID NO: 1-35.

[0100] The term “therapeutically effective” means that the amount ofSH3-BP, for example, which is used, is of sufficient quantity toameliorate the apoptosis associated disorder.

[0101] The subject is e.g., any mammal, e.g., a human, a primate, mouse,rat, dog, cat, cow, horse, pig.

[0102] Also included in the invention also are methods of treatingcell-proliferative disorders associated with JNK activation. The term“cell-proliferative disorder” denotes malignant as well as non-malignantcell populations that often appear to differ morphologically andfunctionally from the surrounding tissue. For example, the method may beuseful in treating malignancies of the various organ systems, in whichactivation of JNK has often been demonstrated, e.g., lung, breast,lymphoid, gastrointestinal, and genito-urinary tract as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer, non-small cell carcinoma of thelung, cancer of the small intestine and cancer of the esophagus. Cancerswith Bcr-Abl oncogenic transformations that clearly require activationof JNK are also included. Essentially, any disorder, which isetiologically linked to JNK kinase activity, would be consideredsusceptible to treatment.

[0103] Methods of Promoting Neuronal Cell Growth and Regeneration

[0104] Also included in the invention are methods promoting (i.e.,increasing) neuronal cell growth or regeneration by contacting a cellwith a SH3 binding peptide, chimeric peptide or nucleic acid of theinvention. For example, the cell is contacted with the peptide of SEQ IDNO: 1-35.

[0105] A neuronal cell is any cell derived from the central orperipheral nervous system, e.g., neuron, neurite or dendrite. The cellpopulation that is exposed to, i.e., contacted with, the SH3 bindingpeptide, fusion peptide or nucleic acid of the invention can be anynumber of cells, i.e., one or more cells, and can be provided in vitro,in vivo, or ex vivo. When the cell is provided in vivo or ex vivo, thesubject may be any mammal, e.g., a human, a primate, mouse, rat, dog,cat, cow, horse, pig.

[0106] Many methods for measuring neuronal cell growth, are known to theskilled artisan including, but not limited to, measuring cell viability.

[0107] For example, to determine whether a compound promotes neuronalcell growth or regeneration, a compound is tested by incubating thecompound with a primary or immortalized neuronal cell inducing a stateof stress of the cells (e.g., by incubating them with H₂O₂ or NMDA, asdescribed herein) and measuring cell viability. As a control the cellsare incubated in the absence if the compound and then the treated cellsare incubated in the absence of the compound and then treated to inducea state of stress. A decrease in cell death (or an increase in thenumber of viable cells) in the compound treated sample indicates thatthe compound promotes cell growth or regeneration.

[0108] Pharmaceutical Compositions

[0109] The SH3 binding peptides, fusion peptides and nucleic acids ofthe invention can be formulated in pharmaceutical compositions. Thesecompositions may comprise, in addition to one of the above substances, apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal orpatch routes.

[0110] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0111] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilizers, buffers, antioxidants and/or other additives may beincluded, as required.

[0112] Whether it is a polypeptide, peptide, or nucleic acid molecule,other pharmaceutically useful compound according to the presentinvention that is to be given to an individual, administration ispreferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0113] Alternatively, targeting therapies may be used to deliver theactive agent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

[0114] Instead of administering these agents directly, they could beproduced in the target cells by expression from an encoding geneintroduced into the cells, e.g. in a viral vector (a variant of theVDEPT technique—see below). The vector could be targeted to the specificcells to be treated, or it could contain regulatory elements, which areswitched on more or less selectively by the target cells.

[0115] Alternatively, the agent could be administered in a precursorform, for conversion to the active form by an activating agent producedin, or targeted to, the cells to be treated. This type of approach issometimes known as ADEPT or VDEPT; the former involving targeting theactivating agent to the cells by conjugation to a cell-specificantibody, while the latter involves producing the activating agent, e.g.an SH3 binding peptide, in a vector by expression from encoding DNA in aviral vector (see for example, EP-A-415731 and WO 90/07936).

[0116] In a specific embodiment of the present invention, nucleic acidsinclude a sequence that encodes an SH3 binding peptide, or functionalderivatives thereof, are administered to modulate activated JNKsignaling pathways by way of gene therapy. In more specific embodiments,a nucleic acid or nucleic acids encoding an SH3 binding peptide, orfunctional derivatives thereof, are administered by way of gene therapy.Gene therapy refers to therapy that is performed by the administrationof a specific nucleic acid to a subject. In this embodiment of thepresent invention, the nucleic acid produces its encoded peptide(s),which then serve to exert a therapeutic effect by modulating function ofthe disease or disorder. Any of the methodologies relating to genetherapy available within the art may be used in the practice of thepresent invention. See e.g., Goldspiel, et al., 1993. Clin Pharm 12:488-505.

[0117] In a preferred embodiment, the Therapeutic comprises a nucleicacid that is part of an expression vector expressing any one or more ofthe αSH3-related peptides, or fragments, derivatives or analogs thereof,within a suitable host. In a specific embodiment, such a nucleic acidpossesses a promoter that is operably-linked to coding region(s) of anSH3 binding peptide. The promoter may be inducible or constitutive, and,optionally, tissue-specific. In another specific embodiment, a nucleicacid molecule is used in which coding sequences (and any other desiredsequences) are flanked by regions that promote homologous recombinationat a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935.

[0118] Delivery of the Therapeutic nucleic acid into a patient may beeither direct (i.e., the patient is directly exposed to the nucleic acidor nucleic acid-containing vector) or indirect (i.e., cells are firsttransformed with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In a specific embodiment of the present invention, anucleic acid is directly administered in vivo, where it is expressed toproduce the encoded product. This may be accomplished by any of numerousmethods known in the art including, e.g., constructing the nucleic acidas part of an appropriate nucleic acid expression vector andadministering the same in a manner such that it becomes intracellular(e.g., by infection using a defective or attenuated retroviral or otherviral vector; see U.S. Pat. No. 4,980,286); directly injecting nakedDNA; using microparticle bombardment (e.g., a “Gene Gun®; Biolistic,DuPont); coating the nucleic acids with lipids; using associatedcell-surface receptors/transfecting agents; encapsulating in liposomes,microparticles, or microcapsules; administering it in linkage to apeptide that is known to enter the nucleus; or by administering it inlinkage to a ligand predisposed to receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can be used to“target” cell types that specifically express the receptors of interest,etc.

[0119] An additional approach to gene therapy in the practice of thepresent invention involves transferring a gene into cells in in vitrotissue culture by such methods as electroporation, lipofection, calciumphosphate-mediated transfection, viral infection, or the like.Generally, the method of transfer includes the concomitant transfer of aselectable marker to the cells. The cells are then placed underselection pressure (e.g., antibiotic resistance) so as to facilitate theisolation of those cells that have taken up, and are expressing, thetransferred gene. Those cells are then delivered to a patient. In aspecific embodiment, prior to the in vivo administration of theresulting recombinant cell, the nucleic acid is introduced into a cellby any method known within the art including, e.g., transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences of interest, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, and similar methodologies that ensure that thenecessary developmental and physiological functions of the recipientcells are not disrupted by the transfer. See e.g. Loeffler and Behr,1993. Meth Enzymol 217: 599-618. The chosen technique should provide forthe stable transfer of the nucleic acid to the cell, such that thenucleic acid is expressible by the cell. Preferably, the transferrednucleic acid is heritable and expressible by the cell progeny.

[0120] In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, e.g., injection of epithelial cells (e.g.,subcutaneously), application of recombinant skin cells as a skin graftonto the patient, and intravenous injection of recombinant blood cells(e.g., hematopoietic stem or progenitor cells). The total amount ofcells that are envisioned for use depend upon the desired effect,patient state, and the like, and may be determined by one skilled withinthe art.

[0121] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

SPECIFIC EXAMPLES Example 1

[0122] Identification of SH3 Binding Peptides

[0123] After determining that the intermediate kinase MKK7 in the JNKsignaling pathway binds to the highly conserved SH3 domains of the IB1and IB2 proteins, it was reasoned an efficient way to block the JNKsignaling pathway would be to prevent the binding of the MKK7 kinase toboth IB1 and IB2. Amino acid sequences important for efficientinteraction with the SH3 domains of IB1 and IB2 were identified bybiopanning a phage display library against GST-SH3_(IB1/2) fusionproteins. The identified amino acid sequences are shown in FIG. 1.Sequence comparison between the sequences obtained during the biopanningexperiment defined two conservative binding motifs SXSVGX (SEQ ID NO. 5)and PPSPRP (SEQ ID NO: 6) (FIG. 1). The latter sequence fits the PXXPSH3 binding consensus (SEQ ID NO: 35). Sequence comparison between thesequences from the biopanning experiment also revealed a consensus αSH3sequence SVSVGMPPSPRP (SEQ ID NO: 1) and a generic αSH3 sequenceSX(S/P)V(G/L)XPPSPRP (SEQ ID NO: 2). As used herein, X may be any aminoacid. The single residue represented by (S/P) may be either Ser or Proin the generic sequence. The single residue represented by (G/L) may beeither Gly or Leu in the generic sequence.

Example 2

[0124] Preparation of SH3 Binding Fusion Proteins

[0125] SH3 binding fusion proteins were synthesized by covalentlylinking the C-terminal end of the αSH3 peptide to a N-terminal 9 aminoacid long carrier peptide derived from the HIV-TAT₄₉₋₅₇ (Vives et al.,J. Biol. Chem. 272: 16010 (1997)). These preparations were designatedTAT (SEQ ID NO: 36) and TAT-αSH3 (SEQ ID NO: 3), respectively. Allretro-inverso TAT-fusion peptides were also synthesized and weredesignated RV-TAT (SEQ ID NO: 38) and RV-TAT-αSH3 (SEQ ID NO: 20),respectively. All D and L peptides were produced by classical F-mocksynthesis and further analyzed by Mass Spectrometry. They were finallypurified by HPLC.

[0126] Generic peptides showing the conserved amino acid residues aregiven in Table 1. An “X” indicates any amino acid. The number of Xs in agiven peptide is not limited to the one depicted, and may vary. Seeabove for a more detailed description of the generic sequences.

Example 3

[0127] Inhibition of MKK7 Binding to IB1 and IB2 By αSH3 Peptide

[0128] Effects of the αSH3 peptide on JNK biological activities werethen studied. Pull-down experiments were used to show that the -αSH3peptide efficiently blocks the binding of MKK7 to both IB1 and IB2 (FIG.2). In the pull-down experiments, the SH3 domains of IB1 and IB2 weresubcloned using PCR into the pGEX-4T1 vector in frame with the GST(Pharmacia). The recombinant proteins were produced in E. Coli.Purification was performed in native conditions using aglutathione-agarose column (Pharmacia). ³⁵S-labelled MKK7 was then usedin classical pull-down experiments.

Example 4

[0129] Inhibition of IL-1β-Induced Pancreatic β-Cell Death By theTAT-αSH3 Peptide

[0130] The effect of the TAT-αSH3 peptide construct on pancreatic β-cellapoptosis induced by IL-1βwas evaluated. Pancreatic βTC-3 cells wereincubated with IL-1β (10 ng/mL) for 48 hours in presence or absence ofthe TAT-αSH3 peptide (FIG. 3). Apoptotic counts were performed followingHoechst/PI staining, as described below. It was determined that theTAT-αSH3 peptide protects insulin-secreting cells against IL-1β-induceddeath.

[0131] The insulin-secreting cell lines INS-1 (Asfari et al., 1992) andβTC-3 (Efrat et al., 1988) were cultured in RPMI 1640 mediumsupplemented with 10% Fetal Calf Serum, 100 μg/ml Streptomycin, 100units/ml Penicillin, 1 mM Na-pyruvate, 2 mM Glutamine and 10 mMβ-mercaptoethanol. TAT, TAT-IB1 and TAT-IB2 peptides were added at aconcentration of 1 μM each 30 minutes prior to the addition of IL-1β (10ng/ml), TNF-α (10 ng/ml) or IFN-γ (100 units/ml). Apoptotic cells werecounted 48 hours after the addition of the cytokines under afluorescence microscope (Axiovert 25, Zeiss) by use of Propidium Iodideand Hoechst 33342 staining (see below). Transfection of cells was donewith “lipofectamin” (Promega).

[0132] The following procedure was used to ensure an accurate measure ofapoptotic β-cells. Pancreatic β-cells in cultures were prepared exactlyas described in details by Hoorens et al. in order to minimizepancreatic β-cells necrosis (Hoorens et al., 1996). Optical microscopywas used following staining of the cells with Hoechst 33342 (HO 342) andpropidium iodide (PI) using conditions optimized for β-cells aspreviously described (Ammendrup et al., 2000; Hoorens et al., 1996).Using this combination of staining, viable or necrotic cells had intactnuclei with, respectively blue (HO 342) or yellow (HO 342 plus PI)fluorescence. Apoptic cells had fragmented nuclei with either a blue (HO342) or yellow (HO 342 plus PI) fluorescence depending on the stage inthe process. For the optical microscopic assays, a minimum of 1000 cellswere counted for each condition. Percentages of living, necrotic andapoptotic cells were then expressed.

Example 5

[0133] Inhibition of Apoptosis in Neurons by the TAT-αSH3 Peptide

[0134] Developing rat cortical neurons were incubated with eitherJNKI_((1 μM)) (FIGS. 4A-4C) or TAT-αSH3_((1 μM)) (FIGS. 4D-4F). Whencompared with control developing rat cortical neurons (FIG. 4A), JNKIwas found to be toxic on the developing neurons, as evidenced by theappearance of residual necrotic bodies (indicated by arrows) (FIGS.4B-4C). The neurons incubated in the presence of TAT-αSH3_((1 μM))lacked residual necrotic bodies (FIGS. 4E-4F), when compared to controldeveloping rat cortical neurons (FIG. 4C).

[0135] It was also found that the TAT-αSH3 peptide (FIG. 4E)reproducibly increased the number of developing neurons obtained overcontrols (FIG. 4D).

Example 6

[0136] Inhibition of NMDA-Induced Apoptosis in Neurons by the TAT-αSH3Peptide

[0137] Rat cortical neurons in culture were incubated withN-methyl-D-aspartate (NMDA), either with or without theTAT-αSH3_((1 μM)) peptide. The cells were then stained with eitherHoechst/PI (FIG. 5A), or with lactate dehydrogenase (LDH). The level ofLDH released in the medium was measured to quantify cell death (FIG.5B). It was found that the TAT-αSH3 peptide completely protected neuronsagainst NMDA-induced death.

Example 7

[0138] In Vitro Solid Phase JNK Assays

[0139] βTC-3 and other insulin-secreting cells will be activated bycytokine treatment before being used for cell extract preparation andprocessed as described (Bonny et al., 2000). Briefly, cellular extractswill be prepared by scraping control and activated cells in lysis buffer(20 mM Tris-acetate, 1 mM EGTA, 1% Triton X-100, 10 mMp-nitrophenyl-phosphate (pNPP), 5 mM sodium pyrophosphate, 10 mMβ-glycerophosphate, 1 mM dithiothreitol). Debris will be removed bycentrifugation for 5′ at 15′000 rpm in an SS-34 rotor (Beckman). 100 μgextracts will be incubated for 1 hour at 4° C. with 1 μg GST-Jun (aminoacids 1-89) or other GST-fusion proteins and 10 μl ofgluthathione-agarose beads (Sigma). Following 4 washes with the scrapingbuffer, the beads will be resuspended in the same buffer supplementedwith 10 mM MgCL₂ and 5 μCiγ³³P-ATP and incubated for 30 minutes at 30°C. Reaction products will then be separated by SDS-PAGE on a denaturing12% polyacrylamide gel. The gels will be dried and subsequently exposedto X-Ray films (Kodak).

[0140] Recombinant JNKs will be produced in a reticulocytetranscription/translation system (Promega). The relevant domains ofbcl-2, bcl-x_(L), p53, c-myc, PPARγ, tau and IRS-1 will be subclonedusing PCR into the pGEX-4T1 vector in frame with the GST (Pharmacia).The recombinant proteins will be produced in E. coli and purified innative conditions using a glutathione-agarose column (Pharmacia). Kinaseassays will be performed by mixing the recombinant JNKs andGST-substrates as described in details (Bonny et al., 2001).

Example 8

[0141] Synthesis of an All-D-retro-inverso Peptides

[0142] Peptides of the invention may be all-D amino acid peptidessynthesized in reverse to prevent natural proteolysis (i.e.,all-D-retro-inverso peptides). An all-D retro-inverso peptide of theinvention would provide a peptide with functional properties similar tothe native peptide, wherein the side groups of the component amino acidswould correspond to the native peptide alignment, but would retain aprotease resistant backbone.

[0143] Retro-inverso peptides of the invention are analogs synthesizedusing D-amino acids by attaching the amino acids in a peptide chain suchthat the sequence of amino acids in the retro-inverso peptide analog isexactly opposite of that in the selected peptide which serves as themodel. To illustrate, if the naturally occurring TAT protein (formed ofL-amino acids) has the sequence RKKRRQRRR (SEQ ID NO: 36), theretro-inverso peptide analog of this peptide (formed of D-amino acids)would have the sequence RRRQRRKKR (SEQ ID NO: 38). The procedures forsynthesizing a chain of D-amino acids to form the retro-inverso peptidesare known in the art. See, e.g., Jameson et al., Nature, 368, 744-746(1994); Brady et al., Nature, 368, 692-693 (1994)); Guichard et al., J.Med. Chem. 39, 2030-2039 (1996). Specifically, the retro-peptides areproduced by classical F-mock synthesis and further analyzed by MassSpectrometry. They are finally purified by HPLC.

[0144] Since an inherent problem with native peptides is degradation bynatural proteases and inherent immunogenicity, the heterobivalent orheteromultivalent compounds of this invention will be prepared toinclude the “retro-inverso isomer” of the desired peptide. Protectingthe peptide from natural proteolysis should therefore increase theeffectiveness of the specific heterobivalent or heteromultivalentcompound, both by prolonging half-life and decreasing the extent of theimmune response aimed at actively destroying the peptides.

OTHER EMBODIMENTS

[0145] While the invention has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

1 40 1 12 PRT Artificial Sequence chemically synthesized 1 Ser Val SerVal Gly Met Pro Pro Ser Pro Arg Pro 1 5 10 2 12 PRT Artificial SequenceVARIANT (2) wherein Xaa is any amino acid 2 Ser Xaa Xaa Val Xaa Xaa ProPro Ser Pro Arg Pro 1 5 10 3 21 PRT Artificial Sequence chemicallysynthesized 3 Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser Val Ser Val GlyMet Pro 1 5 10 15 Pro Ser Pro Arg Pro 20 4 29 PRT Artificial SequenceVARIANT (1)..(4) wherein Xaa is any amino acid 4 Xaa Xaa Xaa Xaa Arg LysLys Arg Arg Gln Arg Arg Arg Xaa Xaa Xaa 1 5 10 15 Xaa Ser Xaa Xaa ValXaa Xaa Pro Pro Ser Pro Arg Pro 20 25 5 6 PRT Artificial SequenceVARIANT (2) wherein Xaa is any amino acid 5 Ser Xaa Ser Val Gly Xaa 1 56 6 PRT Artificial Sequence chemically synthesized 6 Pro Pro Ser Pro ArgPro 1 5 7 12 PRT Artificial Sequence chemically synthesized 7 Ser ValSer Val Gly Met Lys Pro Ser Pro Arg Pro 1 5 10 8 12 PRT ArtificialSequence chemically synthesized 8 Ser Val Ser Val Gly Lys Asn Pro SerPro Arg His 1 5 10 9 12 PRT Artificial Sequence chemically synthesized 9Thr Gln Pro Met Met Ala Pro Pro Ser Pro Arg Gln 1 5 10 10 12 PRTArtificial Sequence chemically synthesized 10 Leu Asp Ser Leu Cys HisPro Gln Ser Pro Arg Pro 1 5 10 11 11 PRT Artificial Sequence chemicallysynthesized 11 His Pro Phe Leu Val Ser Ser Ser Pro Arg Pro 1 5 10 12 9PRT Artificial Sequence chemically synthesized 12 Gly Gln Pro Phe PheSer Pro Phe Ser 1 5 13 11 PRT Artificial Sequence chemically synthesized13 Pro Pro Ser Asn Leu Ile Pro Pro Thr Leu Arg 1 5 10 14 6 PRTArtificial Sequence chemically synthesized 14 Ser Pro Pro Ser Asn Leu 15 15 11 PRT Artificial Sequence chemically synthesized 15 Phe Asn ProTrp Ser Ser Lys Pro Ser Leu Leu 1 5 10 16 12 PRT Artificial Sequencechemically synthesized 16 Asn Ala Ser Val Gly Asn Asp His Ser His SerHis 1 5 10 17 11 PRT Artificial Sequence chemically synthesized 17 GluHis Met Ala Leu Thr Tyr Pro Phe Arg Pro 1 5 10 18 12 PRT ArtificialSequence chemically synthesized 18 Pro Arg Pro Ser Pro Pro Met Gly ValSer Val Ser 1 5 10 19 12 PRT Artificial Sequence VARIANT (7) wherein Xaais any amino acid 19 Pro Arg Pro Ser Pro Pro Xaa Xaa Val Xaa Xaa Ser 1 510 20 21 PRT Artificial Sequence chemically synthesized 20 Pro Arg ProSer Pro Pro Met Gly Val Ser Val Ser Arg Arg Arg Gln 1 5 10 15 Arg ArgLys Lys Arg 20 21 29 PRT Artificial Sequence VARIANT (7) wherein Xaa isany amino acid 21 Pro Arg Pro Ser Pro Pro Xaa Xaa Val Xaa Xaa Ser XaaXaa Xaa Xaa 1 5 10 15 Arg Arg Arg Gln Arg Arg Lys Lys Arg Xaa Xaa XaaXaa 20 25 22 6 PRT Artificial Sequence VARIANT (1) wherein Xaa is anyamino acid 22 Xaa Gly Val Ser Xaa Ser 1 5 23 6 PRT Artificial Sequencechemically synthesized 23 Pro Arg Pro Ser Pro Pro 1 5 24 12 PRTArtificial Sequence chemically synthesized 24 Pro Arg Pro Ser Pro LysMet Gly Val Ser Val Ser 1 5 10 25 12 PRT Artificial Sequence chemicallysynthesized 25 His Arg Pro Ser Pro Asn Lys Gly Val Ser Val Ser 1 5 10 2612 PRT Artificial Sequence chemically synthesized 26 Gln Arg Pro Ser ProPro Ala Met Met Pro Gln Thr 1 5 10 27 12 PRT Artificial Sequencechemically synthesized 27 Pro Arg Pro Ser Gln Pro His Cys Leu Ser AspLeu 1 5 10 28 11 PRT Artificial Sequence chemically synthesized 28 ProArg Pro Ser Ser Ser Val Leu Phe Pro His 1 5 10 29 9 PRT ArtificialSequence chemically synthesized 29 Ser Phe Pro Ser Phe Phe Pro Gln Gly 15 30 11 PRT Artificial Sequence chemically synthesized 30 Arg Leu ThrPro Pro Ile Leu Asn Ser Pro Pro 1 5 10 31 6 PRT Artificial Sequencechemically synthesized 31 Leu Asn Ser Pro Pro Ser 1 5 32 11 PRTArtificial Sequence chemically synthesized 32 Leu Leu Ser Pro Lys SerSer Trp Pro Asn Phe 1 5 10 33 12 PRT Artificial Sequence chemicallysynthesized 33 His Ser His Ser His Asp Asn Gly Val Ser Ala Asn 1 5 10 3411 PRT Artificial Sequence chemically synthesized 34 Pro Arg Phe Pro TyrThr Leu Ala Met His Glu 1 5 10 35 4 PRT Artificial Sequence VARIANT(2)..(3) wherein Xaa is any amino acid 35 Pro Xaa Xaa Pro 1 36 11 PRTArtificial Sequence chemically synthesized 36 Xaa Arg Lys Lys Arg ArgGln Arg Arg Arg Xaa 1 5 10 37 11 PRT Artificial Sequence VARIANT (1)wherein Xaa is any amino acid and Xaa can represent any number of aminoacid residues, including zero 37 Xaa Arg Lys Lys Arg Arg Gln Arg Arg ArgXaa 1 5 10 38 11 PRT Artificial Sequence VARIANT (1) wherein Xaa is anyamino acid and Xaa can represent any number of amino acid residues,including zero 38 Xaa Arg Arg Arg Gln Arg Arg Lys Lys Arg Xaa 1 5 10 3911 PRT Artificial Sequence VARIANT (1) wherein Xaa is any amino acid andXaa can represent any number of amino acid residues, including zero 39Xaa Arg Arg Arg Gln Arg Arg Lys Lys Arg Xaa 1 5 10 40 56 PRT ArtificialSequence chemically synthesized 40 Pro Lys Val Val Ala Leu Tyr Asp TyrGln Ala Arg Glu Ser Asp Glu 1 5 10 15 Leu Ser Phe Lys Lys Gly Asp IleIle Ile Val Leu Glu Lys Ser Asp 20 25 30 Asp Gly Trp Trp Lys Gly Arg LeuLys Gly Thr Lys Glu Gly Leu Ile 35 40 45 Pro Ser Asn Tyr Val Glu Pro Val50 55

What is claimed is:
 1. A peptide comprising the amino acid sequence ofSEQ ID NO:
 2. 2. The peptide of claim 1, wherein said peptide comprisesthe amino acid sequence of SEQ ID NO:
 1. 3. The peptide of claim 1,wherein said peptide binds an islet-brain protein (IB) polypeptide. 4.The peptide of claim 3, wherein said IB polypeptide is IB1 or IB2. 5.The peptide of claim 1, wherein said peptide inhibits MKK7 kinasebinding to an SH3 domain polypeptide.
 6. The peptide of claim 1, whereinsaid peptide comprises D-enantiomeric amino acids.
 7. The peptide ofclaim 1, wherein said peptide is less than 50 amino acids in length. 8.A chimeric peptide comprising a first domain and a second domain linkedby a covalent bond, wherein said first domain comprises the amino acidsequence of SEQ ID NO: 36 and the second domain comprises an SH3 bindingpeptide.
 9. The peptide of claim 8, wherein said SH3 binding peptide isselected from the group consisting of SEQ ID NO: 1-34.
 10. The peptideof claim 8, wherein said SH3 binding peptide binds an islet-brainprotein (IB) polypeptide.
 11. A peptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 7-17.
 12. Thepeptide of claim 11, wherein said peptide binds an islet-brain protein(IB) polypeptide.
 13. The peptide of claim 12, wherein said IBpolypeptide is IB1 or IB2.
 14. The peptide of claim 11, wherein saidpeptide inhibits MKK7 kinase binding to an SH3 domain polypeptide. 15.The peptide of claim 11, wherein said peptide comprises D-enantiomericamino acids.
 16. The peptide of claim 11, wherein said peptide is lessthan 50 amino acids in length.
 17. A peptide less than 50 amino acids inlength comprising (a) an SXSVGX (SEQ ID NO: 5) motif and; (b) a PPSPRP(SEQ ID NO: 6) motif, wherein said peptide binds an SH3 domainpolypeptide.
 18. The peptide of claim 17, wherein said SH3 domainpolypeptide is an islet-brain protein (IB) polypeptide.
 19. The peptideof claim 17, further comprising the amino acid sequence of SEQ ID NO:36.
 20. A peptide comprising the amino acid sequence of SEQ ID NO: 3.21. An isolated nucleic acid encoding the peptide of claim
 1. 22. Avector comprising the nucleic acid of claim
 21. 23. A cell comprisingthe vector of claim
 21. 24. A composition comprising the peptide ofclaim 1 and a carrier.
 25. A method of inhibiting apoptosis in a cell,comprising contacting said cell with the peptide of claim
 1. 26. Themethod of claim 25, wherein said cell is a neuronal cell or a pancreaticcell.
 27. The method of claim 25, wherein said cell is provided invitro, in vivo or ex vivo.
 28. A method of alleviating a symptom of anapoptosis-associated disorder in a subject, said method comprisingadministering to said subject the polypeptide of claim
 1. 29. The methodof claim 28, wherein said apoptosis-associated disorder is selected fromthe group consisting of a neurological disorder, a neurodegenerativedisorder, and a pancreatic disorder.
 30. A method of promoting neuronalcell growth or regeneration, comprising contacting said cell with thepeptide of claim 1.